Regulation of column temperature in liquid chromatography (LC) is less common than in gas chromatography (GC), nevertheless temperature is an important, yet often neglected, separation parameter. Precise control of column temperature can, and has been used to manipulate run times, affect peak efficiency and resolution, increase analyte signal to noise ratios, and even reduce mobile phase solvent consumption, the latter being currently of interest in the area of “green chromatography”. Numerous studies have reported these advantages when using elevated column temperatures in high-performance liquid chromatography/high-pressure liquid chromatography (HPLC), for various modes of liquid chromatography.
Separations carried out at high temperatures also provide the opportunity to apply higher flow rates without the usual increased pressure penalty, due to the decreased viscosity of the mobile phase. The combination of the above mentioned decrease in retention (particularly in reversed-phase mode) and the ability to apply higher than usual flow rates, mean that high temperature separations can achieve tremendous reductions in analysis times, compared to separations carried out at room temperature, up to 50 times faster in some instances. This is particularly relevant in the move towards fast LC and GC, so fast separations are important. Current ovens cannot provide the heating rates required for use with fast separations and so are completely useless when it comes to fast LC.
Typically the known full temperature operating region of high temperature HPLC could perhaps be defined as extending from 60° C. to 374° C., since many of the commonly used solvents in reversed-phase separations would otherwise boil at approximately 65° C., and 374° C. is the highest critical temperature observed for water. Of course there are many techniques that go beyond these limits, such as those that rely on the use of super-critically heated water as the mobile phase, or those at the at the other end of the temperature scale, including some approaches which utilize column cooling below 0° C. In all cases, performing HPLC at non-ambient temperatures requires an accurate, precise and well regulated control of temperature during the separation process, this being particularly important where temperature gradients are being applied. However, it is clear that the task of precise temperature control throughout the column in HPLC is far from trivial, and achieving such temperature precision with rapid gradients generates more difficulty. For example, in addition to “programmed” or intentional applied thermal profiles, there may also be unintentional heating within the column from frictional forces or other causes of non-uniform temperature profiles, which may even cause band broadening and loss of efficiency.
With most modern HPLC systems, various difficulties in obtaining precise and responsive control of column temperature can arise. These are primarily dependent upon type of column oven, but also upon the column dimensions, stationary phase parameters, eluent flow rate and fluid pre-heaters etc. In many instances, the column heater set-point may differ from the actual column temperature by several degrees and temperature variation can occur internally along the length of the column oven, if mobile phase temperature is significantly different to that of the column. In addition, flow rates generating large pressure drops along the column have been suggested to generate frictional heating, especially at pressures above 600 bar, where such heating can be dramatic. In some cases, longitudinal temperature gradients within the column may increase to the point that the column outlet temperature can be over 10° C. higher than the column inlet.
To overcome the problem of temperature differentials at the column inlet, it is known that when elevated temperatures are utilised for the separation, the temperature of the incoming mobile phase should be within ±6° C. of the oven/column temperature, to minimize any band broadening resulting from radial temperature gradients.
It is known to control the temperature of the column in various ways: heating blocks, water jackets and baths, as well as the common circulating air ovens. Conventionally, heaters based upon water jackets and baths have been found to be the most efficient, due to their superior heat capacity. However, such column heaters generally exhibit a rather limited temperature range (though water can be replaced with other liquids with greater heat capacity, for temperatures greater than 100° C.), and a prohibitively slow rate of heating and cooling, in applications where any form of temperature gradient is required. In the case of heating blocks, performance strongly depends on the degree of contact with the column, and in commercial examples where this close contact is maintained, these type of heaters are generally efficient in heat transfer, and also exhibit reasonable heating rates, of approximately 20-30° C. min−1. Circulating air ovens have a heating capacity that depends on the heating rate of the air and speed at which this heated air can be circulated around the column. In general, circulating air based ovens are mainly suitable for isothermal operation. As with liquid bath ovens, and most heating block ovens, circulating air ovens are very limited in their heating and cooling rates, typically <10° C. min−1. In each of the above cases, it can be argued that current performance levels are unsuitable for many fast HPLC applications, particularly those which may require rapid thermal equilibration, such as cases where the application of rapid temperature gradients over short periods is required.
Very few commercially available column ovens are capable of heating beyond 80° C., while fewer still are capable of cooling below 10° C. Gas chromatographic ovens have been used to achieve temperatures as high as 350° C., for ultra-high temperature HPLC; however this approach also has obvious practical limitations, again including relatively slow heating/cooling rates.
There is therefore a need for a column heating arrangements that could address these and other problems. There is also a need for a column cooling arrangement that could address these and other problems.